Background: Fosfomycin is an old antibiotic that is being reconsidered for the treatment of lower urinary tract, due to the global increasing resistance of several bacteria to numerous-antimicrobials. Objectives: To evaluate the activity of fosfomycin and to detect fosfomycin resistant genes (FosA3 and FosC2) in extended-spectrum-beta-lactamase producing Enterobacteriaceae isolates from Menoufia University Hospitals. Methodology: The study included 155 samples collected from patients from different departments and ICUs at Menoufia University Hospitals, detected the ESBL-producing,by combined confirmatory method, fosfomycin resistance was assessed by disk diffusion and the minimal inhibitory concentration (MIC) by E-test method and fosfomycin resistance genes (FosA3 and FosC2) were detected by the conventional PCR. Results: Among the 48 ESBL-producing Enterobacteriaceae isolates analyzed in this study, 21 isolates had fosfomycin resistant genes (FosA3 and FosC2). Conclusion: Fosfomycin showed good antimicrobial activity against multidrug-resistant ESBL-positive Enterobacteriaceae. Resistance genes (FosA3 and FosC2) were detected in some strains, however, other resistance mechanisms to fosfomycin may exist.